No description available.

Communication between epithelial and immune cells is critical for the maintenance and repair of mucosal tissues, with dysregulated epithelia contributing to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, cancer and pulmonary fibrosis. While the role of immune cells in regulating epithelial maintenance and repair has been extensively studied in the distal lung, relatively little is known about the immune microenvironment in the proximal conducting airways, including the function of these immune cells in epithelial regeneration and repair. To study the biology of the human conducting airways, we utilized the mouse trachea as a model tissue and sought to characterize the global immune landscape at homeostasis by multi-parameter flow cytometry and single cell RNA sequencing (scRNA-seq). We then utilized a well-characterized model of tracheal epithelial injury to study changes in the immune landscape in response to injury. These studies revealed that neutrophils are early responders to airway epithelial injury and may function to phagocytose epithelial cell debris. Monocytes and macrophages are then recruited to the injured airway and adopt an activated anti-inflammatory phenotype to participate in tissue repair. Finally, we examined the impact of severe combined immunodeficiency on epithelial cells at homeostasis and following injury. Airway basal stem cells in immunodeficient animals have altered expression of genes related to cytoskeletal support, epithelial adhesion and critical signaling pathways that may impact airway epithelial morphology, barrier integrity, and proliferation and differentiation following injury. Together, these data suggest a functional tracheal immune microenvironment is critical for both the normal development and functional regeneration of the airway epithelium. Macrophages are heterogenous and adaptable immune cells that have has important functions in pulmonary homeostatic maintenance and tissue repair. Distinct subtypes of macrophages have important implications for injury response and repair in the lungs, though relatively little is known about the phenotypes and roles of macrophages in the proximal conducting airways. To address this gap, we characterized murine tracheal macrophages relative to more distal pulmonary macrophages using scRNA-seq and flow cytometry. Tracheal macrophages have a cell surface signature distinct from any previously characterized pulmonary macrophage subtype and were shown to be largely monocyte-derived macrophages generated via fetal liver kinase-2 (Flk2)-dependent adult hematopoiesis. Following polidocanol airway injury, these specialized monocyte-derived tracheal macrophages are recruited to the trachea to become pro-regenerative activated macrophages to aid in regeneration and repair. This macrophage injury response is largely dependent on the chemokine receptor CCR2, with CCR2-deficient mice showing decreased tracheal macrophage recruitment and activation, abnormal epithelial morphology, altered proliferation of airway stem cells, and delayed epithelial repair. Overall, this work highlights the importance of tissue-specific injury-responsive macrophages in airway epithelial regeneration and repair. / 2025-02-12T00:00:00Z

No description available.

No description available.

No description available.

No description available.

BACKGROUND: Despite aggressive dietary modification, lipid lowering medications, and other medical therapy, vascular proliferative diseases continue to account for 50% of all mortality in the United States. It is a significant medical and socioeconomic problem contributing to the mortality of multiple diseases including myocardial infarction (MI), stroke, renal failure, and peripheral vascular disease. With a growing number of children becoming obese and an increase in the number of patients with co-morbidities such as metabolic syndrome and Type 2 diabetes mellitus, epidemiological studies project the morbidity and mortality of these diseases to increase. Among these vascular proliferative diseases are primary atherosclerosis, vascular restenosis, and allograft vasculopathy, all of which are the result of chronic inflammation believed to stem from initial endothelial injury. Once activated by any number of potential injurious agents, endothelial cells (ECs) secrete cytokines that act on multiple cell types. Stimulation of resident vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a normally contractile state to a proliferative state. Following this phenotypic shift, VSMCs migrate from the media to the intima of the artery where they begin secretion of both pro- and anti-inflammatory cytokines. Vascular proliferative disease ensues as a result of the autocrine and paracrine signaling of these cytokines between many different cell types including ECs, VSMCs, macrophages, and T-cells. As a result of the integral role pro- and anti-inflammatory cytokines play in the development of vascular proliferative diseases, they have become the subject of intense study in the field of cardiovascular research. Interleukin-19 (IL-19) is a newly described member of the IL-10 sub-family of anti-inflammatory cytokines. Discovered in 2000, it was originally only thought to be basally expressed in monocytes and lymphocytes, however in 2005 our lab discovered that while uninjured arteries have no detectable IL-19, arteries of patients with vascular proliferative diseases have notable IL-19 expression. Since its discovery in multiple cell types of injured arteries, our lab has subsequently shown that IL-19 inhibits proliferation, migration, spreading, production of reactive oxygen species (ROSs), and expression of pro-inflammatory genes in VSMCs, while in ECs IL-19 has been shown to promote angiogenesis, proliferation, migration, and spreading. AIMS and HYPOTHESIS: The first aim of the current study is to show that IL-19 is expressed in atherosclerotic plaque, and to test that IL-19 can reduce experimental atherosclerosis in susceptible mice. The second aim of the study is to show that IL-19 can regulate development of intimal hyperplasia in a murine model of restenosis. For both aims, we sought to identify potential intracellular signaling mechanisms of IL-19 which produce the observed effect. These aims directed our overall hypothesis that the anti-inflammatory properties of IL-19 can attenuate the vascular response to injury in various animal models of vascular proliferative disease. METHODS and RESULTS: The first aim of this dissertation showed that LDLR-/- mice fed an atherogenic diet and injected with either 1.0ng/g/day or 10.0ng/g/day rmIL-19 had significantly less plaque area in the aortic arch compared with controls (p<0.0001). Weight gain and serum lipid levels were not significantly different. IL-19 could halt, but not reverse expansion of existing plaque. Gene expression in splenocytes from IL-19 treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, IFNgamma, IL-1β and IL-12β, and increased expression of GATA3 messenger ribonucleic acid (mRNA). A greater percentage of lymphocytes were Th2 polarized in IL-19 treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated IL-19 treated mice have significantly less macrophage infiltrate compared with controls (p<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured EC, VSMC, and bone marrow-derived macrophages (BMDM) with IL-19 resulted in a significant decrease in chemokine mRNA, and in the mRNA-stability protein HuR. In the second aim of this dissertation we showed that IL-19 attenuates vascular restenosis in response to carotid artery ligation. Carotid artery ligation of hyper-responsive friend leukemia virus B (FVB) wild-type mice injected with 10ng/g/day rIL-19 had significantly lower neointima/media ratio (I/M) compared with phosphate buffered saline (PBS) controls (p=0.006). Conversely, carotid artery of IL-19-/- mice demonstrated significantly higher I/M ratio compared with wild-type mice (p=0.04). Importantly, the increased I/M ratio in the knockout mice could be rescued by injection of 10ng/g/day IL-19 (p=0.04). VSMC explanted from IL-19-/- mice proliferated significantly more rapidly compared with wild-type (p=0.04). Surprisingly, in this model, IL-19 does not modulate adoptive immunity. Rather, addition of IL-19 to cultured wild-type VSMC did not significantly decrease VSMC proliferation, but could rescue proliferation in IL-19-/- VSMC to wild-type levels (p=0.02). IL-19-/- VSMC expressed significantly greater levels of inflammatory mRNA including IL-1β, TNFα, and MCP-1 in response to TNFα stimulation (p<0.01 for all). No polarization of adaptive immunity was noted in these mice. CONCLUSIONS: These data are the first to report that IL-19 is a potent inhibitor of experimental atherosclerosis via diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. In addition, these data are also the first to show that IL-19 plays a previously unrecognized protective role in vascular restenosis. Together, these data suggest IL-19 is both anti-atherogenic and anti-restenotic and may identify IL-19 as a novel therapeutic to limit vascular inflammation. / Physiology

Thesis advisor: Kenneth C. Williams / Thesis advisor: Welkin Johnson / Understanding the persistence of viral reservoirs despite durable antiretroviral therapy (ART) is essential to addressing the challenge of viral clearance and chronic immune activation with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). It had not previously been demonstrated that HIV or SIV- infected macrophage traffic out of the CNS to reseed the periphery, potentially contributing to viral recrudescence. This thesis proposes the central hypothesis that persistent traffic of monocytes and macrophages out of the CNS and subsequent viral reseeding of the periphery plays a key role in viral dissemination, particularly in the context of acquired immunodeficiency syndrome (AIDS), with ART, and following ART interruption. In Chapter 2, utilizing Superparamagnetic iron oxide nanoparticles (SPION) as a novel in vivo method to label CNS macrophages, we demonstrate that under normal conditions, CNS macrophages migrate out to the deep cervical lymph nodes. However, during SIV infection, we observe an accumulation of macrophages within the CNS and a reduction in traffic out to the periphery. Importantly, with SIV-infection, we found that SIV-infected macrophages traffic out to deep cervical lymph nodes. From these, we find that under normal conditions, macrophages traffic out of the CNS. However, during SIVinfection, macrophages are retained within the CNS, contributing to inflammation in the brain, and those that do migrate out are virally infected. In Chapter 3, we hypothesized that ART restores CNS macrophage traffic and prevents viral dissemination from the CNS reservoir by eliminating the traffic of virally infected macrophage out of the CNS, as seen with AIDS and SIV-induced encephalitis (SIVE). We also hypothesized that following four weeks of ART interruption there would be expansion of the CNS viral reservoir with traffic out of virally infected macrophages to the deep cervical lymph node. Utilizing a rapid AIDS model with CD8 depletion to induce a high incidence of SIVE and intracisternal injection of SPION, we found that SIV-infected macrophages accumulate in the perivascular space, meninges, choroid plexus, and traffic out at a low rate to the deep cervical lymph node, spleen, and even to the dorsal root ganglia (DRG). With ART, we found clearance of virally infected macrophages in the brain perivascular space but not in the meninges or choroid plexus. Importantly following four weeks of ART interruption, the perivascular space remained clear of virus but there was a rebound in the meninges and scattered virally infected macrophages in the choroid plexus. With ART and following a brief ART interruption, there was no traffic of CNS virally infected macrophages out to the deep cervical lymph node, spleen, or DRGs. These data demonstrate that ART effectively clears virus-infected perivascular macrophages and eliminates the traffic of virus-infected macrophages out of the CNS to the deep cervical lymph node and spleen but does not eliminate virally infected macrophages in the meninges or choroid plexus. By using two differently colored SPION injected early and late, we observed an increase in early SPION+ macrophages within and outside the CNS with SIVE, ART, and ART interruption, indicating that SIV-infected perivascular macrophages establish an early viral reservoir with ongoing seeding in the meninges and choroid plexus throughout infection. These findings are consistent with the retention of CNS macrophages in the presence of inflammation and viral infection, as well as the potential for viral rebound in the CNS from sources such as the blood, meninges, and choroid plexus with ART and following ART interruption. In Chapter 4, we propose a novel pathway for virus-infected macrophages to traffic out of the CNS via cranial and spinal nerves. Due to the persistence of virally infected macrophages in the meninges with durable ART and continuity of the CNS meninges with peripheral nerves, we hypothesize that virally infected macrophage traffic out of the CNS via cranial and peripheral nerves with AIDS and SIVE, on ART, and following ART interruption. To test this hypothesis, we tracked SPION+ macrophages by quantifying them at central (spinal cord and cranial nerves) and peripheral sites (dorsal root ganglia, DRG). Similar to our previous findings in the brain, SIV infection increased the numbers of macrophages in the spinal cord and decreased them in peripheral sites. Staining for viral RNA and GP41 identified virus-infected SPION+ macrophages in cranial nerves and DRG, which were significantly reduced but not eliminated by ART. In animals with AIDS, late- and dual-labeled SPION+ macrophages decreased, suggesting reduced macrophage trafficking late in infection. ART appeared to restore traffic, as higher numbers of late- and dual-labeled macrophages were observed, though this reversed to levels seen in AIDS/SIVE upon ART interruption. Our findings reveal a previously understudied pathway that allows CNS macrophage viral reservoirs to reseed virus to the periphery, a process that persists despite ART. In Chapter 5, we performed a literature review to better understand the effects of HIV infection on aging, as age is a primary risk factor for the development of HIV-associated neurocognitive disorders and HIV-associated sensory neuropathy. With ART extending the lifespan of people living with HIV, they now also experience accelerated aging, leading to earlier onset of age-related conditions like cardiovascular disease and neurocognitive disorders. Evidence suggests this is due to chronic immune activation, co-infections, and possibly ART itself. HIV and aging both alter immune cell populations, increasing inflammatory markers and contributing to "inflamm-aging." While ART slows this acceleration, it cannot prevent aging or related comorbidities. This thesis explores the role of macrophage traffic from the CNS and its contribution to the spread of the virus to peripheral tissues. To investigate this, we utilized a novel in vivo labeling method to track CNS macrophages, identify migration out of the CNS, and evaluate how ART and ART interruption influence the traffic of virally infected macrophages to peripheral tissues. Our findings underscore the role of CNS macrophages in the resolution of inflammation by trafficking out of the CNS, viral rebound from blood- derived sources following ART interruption, and the role of perineural pathways in viral dissemination even with durable ART. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Pre-pubertal bovine mammary development involves ductal and stromal tissue changes. In mice, this process is impacted by presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. We studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in pre-pubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the cells in formalin fixed, paraffin embedded mammary tissue. The first set (ONT) included samples (n=4/week) from birth to 6 weeks of age. Another set (OVX) determined the influence of ovaries, 19 animals were intact or ovariectomized 30 days before sampling. They were 90, 120 or 150 days old at examination. The third set (EST) allowed examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. Samples were from calves given estrogen implants (n=6) or placebo (n=4) at 56 days old, and sampled at 70 days old. We examined 20 images each of NEAR and FAR stroma from every animal. More eosinophils were observed in NEAR versus FAR in the ONT and OVX , more mast cells observed in NEAR versus FAR in ONT. More macrophages were observed in NEAR versus FAR in ONT and EST. We show, for the first time, that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure. We suggest a possible role for these cells in control of bovine mammary development. / Master of Science

Keratins are fibrous structural proteins found in human hair that have been used to develop bioactive and biocompatible constructs for a wide variety of tissue engineering and healthcare applications. Their ubiquity, capacity for self-assembly, ease of use and versatility in blended materials, and ability to modulate cell behavior and promote tissue ingrowth have made keratins well-suited for the development of regenerative therapies. In particular, keratins have demonstrated bioactivity in both in-vivo and in-vitro studies, by altering immune and stem cell phenotype and function and promoting an anti-inflammatory/wound healing environment. This work seeks to build on previous research by investigating the ability of low and high molecular weight keratins to augment anti-inflammatory primary macrophage phenotypes and examining the influence of keratin biomaterials on cellular and inflammatory mechanisms in two models of injury and disease. Rodent models of blast induced neurotrauma (BINT) and severe osteoporosis were used to inform the development of 2D and 3D in-vitro models of macrophage/endothelial cell injury and osteogenic differentiation respectively. Keratin biomaterials exhibited some potential to alter macrophage and endothelial cell dynamics following blast, specifically by promoting anti-inflammatory (M2c-like) macrophage polarization and diminishing endothelial cell injury responses (i.e. endothelial glycocalyx shedding). A more clinically relevant model of osteoporosis found that stem cells harvested from older, osteoporotic animals demonstrated limited proliferative and bone differentiation potential compared to healthy cells. However, 3D constructs (especially keratin-based materials) were able to enhance calcification and osteogenic gene expression of diseased cells. These results highlight the complexity of macrophage phenotypic switching and cellular dynamics in these systems. However, keratin-based therapeutics may prove useful for facilitating tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease. / Ph. D. / Keratins are proteins found in human hair that have been used for a wide variety of healthcare applications. Their availability, ease of use as coatings, gels, and scaffolds, and their ability to alter cell function have made keratins well-suited for regenerative therapies. In particular, keratins have demonstrated the ability to alter immune and stem cell function by promoting a wound healing environment. This work seeks to investigate the ability of different keratins to enhance wound healing immune cell types and examine the influence of keratin materials on stem and blood vessel cell behavior in two models of injury and disease. Rodent models of blast-wave induced traumatic brain injury (concussion) and severe osteoporosis (bone brittleness) were used to develop cell culture models of immune cell and blood vessel cell injury as well as the conversion of stem cells to bone-building cells respectively. Keratin-based materials exhibited some potential to alter immune and blood vessel cell function following blast injury, specifically by promoting wound healing immune cell transformation and diminishing blood vessel cell injury responses. A more clinically relevant model of osteoporosis found that stem cells harvested from older animals had a more limited ability to divide and transform to bone cells compared to healthy cells. However, 3D gels (especially keratin-based materials)—unlike 2D coatings—were able to enhance calcium deposition and other bone markers in diseased cell cultures. These results highlight the complexity of cell responses in these systems. However, keratin-based therapeutics may prove useful for promoting tissue regeneration and limiting detrimental inflammatory and cellular responses in various models of injury and disease.